Excitotoxicity is of critical importance in the neuronal death that occurs after epilepsy, stroke, traumatic brain injury, and a range of other CNS pathologies. In epilepsy, the balance between cerebral excitation and inhibition is disrupted, leading to uncontrolled excitability of groups of neurons and subsequent excitotoxic cell death. Brain damage is caused by persistent and highly repetitive seizures that are associated with excitotoxic cell death mechanisms. However, our understanding of the molecular pathways that regulate cell death after seizure activity remain in their infancy and largely lag behind work in other areas of brain injury. We have shown that inbred strains of mice show remarkable genetic differences in susceptibility to excitatory amino acid-induced cell death. During the last four years of present RO1 funding, we have identified the location of three significant quantitative trait loci (QTL) on chromosomes 18, 15, and 4 in the mouse genome, responsible for seizure-induced cell death susceptibility, using genome exclusion mapping with DNA-based markers in a backcross population derived from the C57BL/6 and FVB/N inbred mouse strains. The goal of this renewal application is to identify the genes causing genetic variation in susceptibility to seizure-induced excitotoxic cell death using fine-mapping, positional candidate and positional cloning techniques. To identify the genes that underlie each QTL, we have proposed three specific aims. The congenic mapping studies of Specific Aim 1 will be used to confirm the existence of and fine map these murine cell death susceptibility loci. We will rigorously assess the strength of the provisional genetic mapping assignments, confirm the map positions, and reassess the phenotypic effects of these loci. The fine mapping studies outlined in Specific Aim 2 will narrow each QTL interval down to less than a 1 cM interval using interval-specific congenic strains. This expansion of individuals with an identified donor chromosome will allow us to test each chromosomal segment statistically for linkage to kainate-induced cell death. In Specific Aim 3, we will assess and identify candidate genes in the introgressed regions using two approaches. For gene identification, we will first identify candidate genes by utilizing the rapidly emerging mouse and human genome resources relating to genes and gene maps. Candidate genes will be sought based on known or deduced function with regard to seizure-induced excitotoxic cell death. New and known genes will be identified through strain sequence comparison using single-stranded conformation polymorphism (SSCP) analysis. We will then test for single nucleotide polymorphism (SNP) haplotype among inbred mouse strains for association with susceptibility to seizure-induced cell death and also assess differential expression of candidate genes using RT-PCR and in situ hybridization approaches. Results gleaned from these studies will facilitate our understanding of the biological mechanisms that underlie neuronal sensitivity and provide information regarding the pathogenesis of human epilepsy. [unreadable] [unreadable] [unreadable]